Chitosan-coated liposomes for intracellular oligonucleotides delivery: characteristics and cell uptake behavior

Surface modification of liposomes with polymer to optimize drug delivery was well developed recently. The objective of the present work was to evaluate the feasibility of chitosan-coated liposomes (CSLP) as vehicles for anti-sense oligodeoxynucleotides (ASON). CSLP was obtained by adding chitosan dropwise to liposomes under magnetic stirring. The effect of chitosan content on size, zeta potential, and coating efficiency was investigated, which showed that chitosan increased the size and zeta potential of CSLP, and the coating efficiency increased with chitosan content increasing. Agarose gel electrophoresis was employed to evaluate the loading efficiency of CSLP for ASON, from which one could see ASON was completely combined to CSLP when the mass ratio of total lipids:ASON was more than 50:1. MTT assay showed that CSLP took on very low cytotoxicity, which is much lower than chitosan. At last, cell uptake behavior was investigated by a flow cytometer, which showed that CSLP enhanced significantly the COS7 cells uptake of ASON. All the results indicated that the CSLP could be a promising non-viral ASON vehicle.     

Evaluation of mucoadhesiveness of polymers by BIACORE method and mucin-particle method

To evaluate the reliability of the BIACORE method as a useful method for measuring the mucoadhesive interaction between chitosan and mucin, the mucin-particle method was used for comparison. In this study, the adhesivities of different-molecular-weight chitosans (chitosan Mw. 150,000, CS; low-molecular-weight chitosan, LCS) and hydrophobically modified chitosans (dodecylated CS, d-CS; dodecylated LCS, d-LCS) to mucin were determined. The BIACORE method showed that CS, LCS and d-CS could interact with mucin based on the increased resonance unit (RU) response after mucin was passed over the chitosans-immobilized sensor chip surface. Sensorgrams obtained from the interaction between these polymers and mucin also indicated the rate and strength of binding reaction. The rate and strength were higher for unmodified chitosans than hydrophobically modified chitosans. The simple in vitro mucoadhesive test or mucin-particle method revealed that the turbidity of unmodified chitosan/mucin mixtures was higher than that of dodecylated chitosans for all concentration of chitosans and mucin. The results from both BIACORE and the mucin-particle method implied that hydrophobic modification of chitosan reduced its adhesivity to mucin. The results from these two methods corresponded well. Therefore, the BIACORE method has promised as an alternative method for evaluating the adhesivity of adhesive polymers to mucin.     

Chitosan-coated liposomes for intracellular oligonucleotides delivery: Characteristics and cell uptake behavior

Surface modification of liposomes with polymer to optimize drug delivery was well developed recently. The objective of the present work was to evaluate the feasibility of chitosan-coated liposomes (CSLP) as vehicles for anti-sense oligodeoxynucleotides (ASON). CSLP was obtained by adding chitosan dropwise to liposomes under magnetic stirring. The effect of chitosan content on size, zeta potential, and coating efficiency was investigated, which showed that chitosan increased the size and zeta potential of CSLP, and the coating efficiency increased with chitosan content increasing. Agarose gel electrophoresis was employed to evaluate the loading efficiency of CSLP for ASON, from which one could see ASON was completely combined to CSLP when the mass ratio of total lipids:ASON was more than 50:1. MTT assay showed that CSLP took on very low cytotoxicity, which is much lower than chitosan. At last, cell uptake behavior was investigated by a flow cytometer, which showed that CSLP enhanced significantly the COS7 cells uptake of ASON. All the results indicated that the CSLP could be a promising non-viral ASON vehicle.     

Novel mucoadhesion tests for polymers and polymer-coated particles to design optimal mucoadhesive drug delivery systems

To design an effective particulate drug delivery system having mucoadhesive function, several mucoadhesion tests for polymers and the resultant particulate systems were developed. Mucin particle method is a simple mucoadhesion test for polymers, in which the commercial mucin particles are used. By measuring the change in particle size or zeta potential of the mucin particle in a certain concentration of polymer solution, we could estimate the extent of their mucoadhesive property. BIACORE method is also a novel mucoadhesion test for polymers. On passing through the mucin suspension on the polymer-immobilized chip of BIACORE instrument, the interaction was quantitatively evaluated with the change in its response diagram. By using these mucoadhesion tests, we detected a strong mucoadhesive property of several types of chitosan and Carbopol. Evaluation of mucoadhesive property of polymer-coated particulate systems was demonstrated with the particle counting method developed by us. To detect the mucoadhesive phenomena in the intestinal tract, we observed the rat intestine with the confocal laser scanning microscope (CLSM) after oral administration of the particulate systems. The resultant photographs clearly showed a longer retention of submicron-sized chitosan-coated liposomes (ssCS-Lip) in the intestinal tract than other liposomal particles tested such as non-coated liposomes and chitosan-coated multilamellar one. These observations explained well the superiority of the ssCS-Lip as drug carrier in oral administration of calcitonin in rats than other liposomal particles.     

The effect of particle structure of chitosan-coated liposomes and type of chitosan on oral delivery of calcitonin

To optimize the properties of chitosan-coated liposomes for oral administration of peptide drugs, we examined the effect of type of chitosan and the structure of liposomal systems on the mucoadhesiveness of liposomes and resultant pharmacological effects of the liposomal peptide drug. A low-molecular weight chitosan (LCS) and a high-molecular weight chitosan (CS) were used as coating polymers of liposomes containing elcatonin (eCT). The muco-penetrative behaviors across the mucous gel layer covering the intestinal epithelial cells and the pharmacological effect after intragastric administration were determined in rats. The results showed that both LCS-coated liposomes (LCS-Lips) and CS-coated liposomes (CS-Lips) could permeate the mucous layer in the small intestine. The most interesting result was that LCS-Lips containing eCT showed remarkably more prolonged effectiveness in decreasing the blood calcium concentration than did CS-Lips containing eCT, moreover, it was also found that LCS had more efficiency to protect eCT from the enzymatic degradation than CS. In comparing the area above the plasma calcium concentration time curves (AAC) values among eCT-containing liposomes with different structures, i.e. eCT adsorbed on coated liposomes (eCT-ad-CS-Lip, eCT-ad-LCS-Lips) and eCT encapsulated in coated liposomes (eCT-encap-CS-Lips, eCT-encap-LCS-Lips), eCT-encap-CS-Lip showed much higher effectiveness than eCT-ad-CS-Lip. However, the AAC value for eCT-ad-LCS-Lip was comparable to that for eCT-encap-CS-Lip, while the value for eCT-ad-CS-Lip was nearly zero. These results suggested that LCS is a good mucoadhesive polymer candidate for enhancing the bioavailability of orally administered peptide containing liposomes, while encapsulation of eCT within the liposomal particles is important to protect eCT against enzymatic degradation in the gastrointestinal (GI) tract.     

Effectiveness of submicron-sized, chitosan-coated liposomes in oral administration of peptide drugs

The mucoadhesive behavior of chitosan-coated liposomes in the intestinal tract of the rat was examined to elucidate their particle size effects on the absorption of an entrapped drug, calcitonin. The intestine was removed from rats after oral administration of liposomes containing a fluorescent dye, and its various parts were observed with confocal laser scanning microscopy. Penetration of submicron-sized liposomes (ssLip) or chitosan-coated ssLip (ssCS-Lip) into the mucosa was observed, while such behavior was not observed for the multilamellar liposomes, even when coated with chitosan (CS-Lip). The retentive property of ssCS-Lip was confirmed by measuring the amount of dye in each part of the intestine. The pharmacologic effects of calcitonin-loaded liposomes of different particle size were measured after oral administration in rats. The pharmacologic effect of oral administration of ssLip coated with chitosan was detected up to 120 h after administration. The extensive pharmacologic effect of ssCS-Lip was attributed to their prolonged retention in the intestinal mucosa, partly owing to their penetrative property into the intestinal mucosa. The chitosan-coated ssLip, with their higher retentive property in the intestinal tract, are much more effective than ssLip and CS-Lip in improving the enteral absorption of peptide drugs.     

Hypoglycemic efficacy of chitosan-coated insulin liposomes after oral administration in mice

INTRODUCTION In the past two decades the potential usefulnessof liposomes as drug carriers for improving enteral ab-sorption of poorly absorbed drugs including peptidedrugs such as insulin has attracted considerable interest.These phospholipid vesicles are capable of encapsulat-ing both hydrophobic and hydrophilic drugs; they arebiodegradable and are not toxic in vivo. The drugsencapsulated in liposomes are sufficiently protectedfrom enzymatic attack and immune recognition[1]. Li-po…     

Mucoadhesive chitosan-coated liposomes: characteristics and stability

Lecithin liposomes, empty or containing FITC-dextran, were prepared by the ethanol injection method. Three different types of chitosans with different molecular weight and degrees of deacetylation were used (Seacure 113, 210 and 311). Chitosan coating was carried out by mixing the liposomal suspension with the chitosan solution followed by incubation. The size of liposomes was measured before and after polymer coating by an image analysis technique. The mean diameter of liposomes containing FITC-dextran was in the size range 250-280nm, whereas the size after coating was 300-330nm, regardless of chitosan type. All chitosan-coated liposomes were of spherical shape and no morphological differences between uncoated and coated liposomes were observed. Liposomes with FITC-dextran, originally entrapping 50% of the marker substance taken in the preparation and coated in the presence of unentrapped marker substance, contained 60-65%of the marker substance. The highest entrapment was found for liposomes coated with medium molecular weight chitosan. The stability of chitosan-coated liposomes in simulated gastric fluid was significantly higher as compared to uncoated liposomes. One can conclude that chitosan is stabilizing the original liposomal structure and protecting liposomally entrapped drug.     

Mucoadhesive chitosan-coated liposomes: characteristics and stability

Lecithin liposomes, empty or containing FITC-dextran, were prepared by the ethanol injection method. Three different types of chitosans with different molecular weight and degrees of deacetylation were used (Seacure 113, 210 and 311). Chitosan coating was carried out by mixing the liposomal suspension with the chitosan solution followed by incubation. The size of liposomes was measured before and after polymer coating by an image analysis technique. The mean diameter of liposomes containing FITC-dextran was in the size range 250-280 nm, whereas the size after coating was 300-330 nm, regardless of chitosan type. All chitosan-coated liposomes were of spherical shape and no morphological differences between uncoated and coated liposomes were observed. Liposomes with FITC-dextran, originally entrapping 50% of the marker substance taken in the preparation and coated in the presence of unentrapped marker substance, contained 60-65% of the marker substance. The highest entrapment was found for liposomes coated with medium molecular weight chitosan. The stability of chitosan-coated liposomes in simulated gastric fluid was significantly higher as compared to uncoated liposomes. One can conclude that chitosan is stabilizing the original liposomal structure and protecting liposomally entrapped drug.     

Development and characterization of polymer-coated liposomes for vaginal delivery of sildenafil citrate

Vaginal administration of sildenafil citrate has shown recently to develop efficiently the uterine lining with subsequent successful embryo implantation following in vitro fertilization. The aim of the present study was to develop sildenafil-loaded liposomes coated with bioadhesive polymers for enhanced vaginal retention and improved drug permeation. Three liposomal formulae were prepared by thin-film method using different phospholipid:cholesterol ratios. The optimal liposomal formulation was coated with bioadhesive polymers (chitosan and HPMC). A marked increase in liposomal size and zeta potential was observed for all coated liposomal formulations. HPMC-coated liposomes showed the greater bioadhesion and higher entrapment efficiency than chitosan-coated formulae. The in vitro release studies showed prolonged release of sildenafil from coated liposomes as compared to uncoated liposomes and sildenafil solution. Ex vivo permeation study revealed the enhanced permeation of coated relative to uncoated liposomes. Chitosan-coated formula demonstrated highest drug permeation and was thus selected for further investigations. Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR) confirmed the successful coating of the liposomes by chitosan. Histopathological in vivo testing proved the efficacy of chitosan-coated liposomes to improve blood flow to the vaginal endometrium and to increase endometrial thickness. Chitosan-coated liposomes can be considered as potential novel drug delivery system intended for the vaginal administration of sildenafil, which would prolong system's retention at the vaginal site and enhance the permeation of sildenafil to uterine blood circulation.     

The effect of particle structure of chitosan-coated liposomes and type of chitosan on oral delivery of calcitonin

To optimize the properties of chitosan-coated liposomes for oral administration of peptide drugs, we examined the effect of type of chitosan and the structure of liposomal systems on the mucoadhesiveness of liposomes and resultant pharmacological effects of the liposomal peptide drug. A low-molecular weight chitosan (LCS) and a high-molecular weight chitosan (CS) were used as coating polymers of liposomes containing elcatonin (eCT). The muco-penetrative behaviors across the mucous gel layer covering the intestinal epithelial cells and the pharmacological effect after intragastric administration were determined in rats. The results showed that both LCS-coated liposomes (LCS-Lips) and CS-coated liposomes (CS-Lips) could permeate the mucous layer in the small intestine. The most interesting result was that LCS-Lips containing eCT showed remarkably more prolonged effectiveness in decreasing the blood calcium concentration than did CS-Lips containing eCT, moreover, it was also found that LCS had more efficiency to protect eCT from the enzymatic degradation than CS. In comparing the area above the plasma calcium concentration time curves (AAC) values among eCT-containing liposomes with different structures, i.e. eCT adsorbed on coated liposomes (eCT-ad-CS-Lip, eCT-ad-LCS-Lips) and eCT encapsulated in coated liposomes (eCT-encap-CS-Lips, eCT-encap-LCS-Lips), eCT-encap-CS-Lip showed much higher effectiveness than eCT-ad-CS-Lip. However, the AAC value for eCT-ad-LCS-Lip was comparable to that for eCT-encap-CS-Lip, while the value for eCT-ad-CS-Lip was nearly zero. These results suggested that LCS is a good mucoadhesive polymer candidate for enhancing the bioavailability of orally administered peptide containing liposomes, while encapsulation of eCT within the liposomal particles is important to protect eCT against enzymatic degradation in the gastrointestinal (GI) tract.     

Chitosan-coated liposomes: characterization and interaction with leuprolide

The objective of the present work was to investigate the effect of chitosan concentration and lipid type on the characteristics of chitosan-coated liposomes and their interactions with leuprolide. Liposomes from lipid of high purity and low purity were prepared and coated by chitosan. Physical properties, drug entrapment efficiency, and stability upon dilution were respectively compared. Results showed that the particle size increment of liposomes from low purity lipid was larger than that from high purity lipid, indicating a thicker coating layer. The high zeta potential of particles from low purity lipid was thought to play an important role in the resistance to flocculation. As to particles from high purity lipid, polymer bridging caused flocculation at low polymer concentration while at high concentration, the adsorbed chitosan molecule led to steric stabilization. Drug entrapment efficiency decreased as chitosan was added to liposomes, showing the disturbance of bilayers. Upon dilution, the leakage of leuprolide from low purity liposomes was larger than that from high purity liposomes. In conclusion, low purity lipid possessed more negative charge and formed thicker adsorptive layer by stronger electrostatic attraction with chitosan. The interaction between chitosan and the polar head groups on the surface of phospholipid bilayers may interfere with leuprolide entrapped in liposomes and result in the leakage of leuprolide.     

小鼠口服多糖包覆胰岛素脂质体的降血糖作用小鼠口服多糖包覆胰岛素脂质体的降血糖作用

目的 研究壳聚糖和海藻酸钠两种多糖包覆胰岛素脂质体的小鼠po降血糖作用。方法 用逆相蒸发法制备胰岛素脂质体;用透射电镜和激光粒度仪测定它们的形态和粒径;用HPLC法和超速离心法测定包封率;用胃蛋白酶和胰蛋白酶溶液试验多糖包覆脂质体对胰岛素的保护作用;用酶-苯酚法测定小鼠po多糖包覆胰岛素脂质体后降血糖作用。结果小鼠po 0.1%壳聚糖和0.1%海藻酸钠包覆的胰岛素脂质体具有较好的降血糖作用。结论壳聚糖或海藻酸钠包覆的脂质体能减少胃蛋白酶或胰蛋白酶对胰岛素的降解并促进胰岛素po吸收。     

Evaluation of Mucoadhesive PLGA Microparticles for Nasal Immunization

In this study, hepatitis B surface antigen (HBsAg) loaded poly(lactic-co-glycolic acid) (PLGA) microparticles were prepared and coated with chitosan and trimethyl chitosan (TMC) to evaluate the effect of coating material for nasal vaccine delivery. The developed formulations were characterized for size, zeta potential, entrapment efficiency, and mucin adsorption ability. Plain PLGA microparticles demonstrated negative zeta potential. However, coated microparticles showed higher positive zeta potential. Results indicated that TMC microparticles demonstrated substantially higher mucin adsorption when compared to chitosan-coated microparticles and plain PLGA microparticles. The coated and uncoated microparticles showed deposition in nasal-associated lymphoid tissue under fluorescence microscopy. The coated and uncoated microparticles were then administered intranasally to mice. Immune-adjuvant effect was determined on the basis of specific antibody titer observed in serum and secretions using enzyme-linked immunosorbent assay. It was observed that coated particles showed a markedly increased anti-HBsAg titer as compared to plain PLGA microparticles, but the results were more pronounced with the TMC-coated PLGA microparticles.     

Chitosan dispersed system for colon-specific drug delivery

A chitosan dispersed system (CDS), which was composed of active ingredient reservoir and the outer drug release-regulating layer dispersing chitosan powder in hydrophobic polymer, was newly developed for colon-specific drug delivery. An aminoalkyl methacrylate copolymer RS (Eudragit) RS) was selected as a hydrophobic polymer because it is hardly dissolved in acidic medium in which easily dissolves chitosan. In order to obtain the bi-functional releasing characteristics, i.e. time dependent and site specific, capsules containing the active ingredient (Drug Capsules) were coated by the chitosan dispersed hydrophobic polymer, resulting in CDS Capsules. The release rate could be controlled by changing the thickness of the layer. Furthermore, for colon-specific drug delivery, an additional outer enteric coating was necessary to prevent the drug release from CDS Capsules in the stomach, since chitosan dispersed in the layer dissolves easily under acidic conditions. Resultant enteric-coated CDS Capsules reached the large intestine within 1-3 h after oral administration and they were degraded at the colon in beagle dogs.     

Chitosan–gum arabic polyelectrolyte complex films: physicochemical,mechanical and mucoadhesive properties

By blending chitosan (CS) and gum arabic (GA), a powerful biomaterial complex might be obtained due to the unique properties of CS and the low viscosity and good emulsifying properties of GA. The objectives of this study were to prepare and examine the properties of dispersions and films of CS and GA as a function of the mixing weight ratio, pH value and molecular weight of CS. The dispersions were characterized by turbidity, zeta potential and cytotoxicity and then the dispersions were cast into films. Physicochemical properties of the film were performed. CS–GA dispersions exhibited higher turbidity and a lower zeta potential with an increase in the GA ratio. Continuous films of the CS–GA could be formed at all ratios. CS and GA could molecularly interact via electrostatic forces and intermolecular hydrogen bonding. The CS–GA (1:0.5) films exhibited relatively low water uptake, erosion, water vapor permeability and puncture strength compared to the CS films. Furthermore, the CS–GA films demonstrated good mucoadhesive properties, allowing for adhesion to the mucosal membrane. Based on these results, it could be advantageous to use CS–GA films as film formers for the formulation of coatings and drug delivery systems.     

醋酸亮丙瑞林脂质体及壳聚糖包衣脂质体经肠道及Caco-2细胞转运机制

目的考察醋酸亮丙瑞林脂质体及壳聚糖包衣脂质体经大鼠肠道及Caco-2细胞的转运机制。方法应用翻转肠囊法和Caco-2 细胞模型考察游离醋酸亮丙瑞林、脂质体包封的醋酸亮丙瑞林以及壳聚糖包衣脂质体中醋酸亮丙瑞林的转运特征。在Caco-2细胞水平考察壳聚糖浓度、加入次序对脂质体中醋酸亮丙瑞林渗透的影响。结果游离醋酸亮丙瑞林的转运符合被动扩散的性质。相同实验条件下，脂质体中药物渗透量低于游离药物。可能是由于脂质体包封醋酸亮丙瑞林，阻止了药物向肠囊和Caco-2细胞转运。但是脂质体对酶降解醋酸亮丙瑞林具有保护作用。壳聚糖促进脂质体中亮丙瑞林的转运，壳聚糖的促渗作用在0.1%-0.5%浓度范围无明显差异。壳聚糖包覆脂质体后的促渗作用弱于其单独使用的促渗作用。结论壳聚糖包衣醋酸亮丙瑞林脂质体兼有保护和促渗作用，可能促进醋酸亮丙瑞林的口服吸收。     

Novel O-palmitoylscleroglucan-coated liposomes as drug carriers: development, characterization and interaction with leuprolide

Polysaccharide-coated liposomes have been studied for their potential use for peptide drug delivery by the oral route because they are able to minimize the disruptive influences on peptide drugs of gastrointestinal fluids. The aim of this work was to synthesize and characterize a modified polysaccharide, O-palmitoylscleroglucan (PSCG), and to coat unilamellar liposomes for oral delivery of peptide drugs. To better evaluate the coating efficiency of PSCG, also scleroglucan (SCG)-coated liposomes were prepared. We studied the surface modification of liposomes and the SCG- and PSCG-coated liposomes were characterized in terms of size, shape, zeta potential, influence of polymer coating on bilayer fluidity, stability in serum, in simulated gastric and intestinal fluids and against sodium cholate and pancreatin. Leuprolide, a synthetic superpotent agonist of luteinizing hormone releasing hormone (LHRH) receptor, was chosen as a model peptide drug. After polymer coating the vesicle dimensions increased and the zeta potential shifted to less negative values. These results indicate that both SCG- and PSCG-coated liposomes surface and DSC results showed that PSCG was anchored on the liposomal surface. The stability of coated-liposomes in SGF, sodium cholate solution and pancreatin solution was increased. From this preliminary in vitro studies, it seems that PSCG-coated liposomes could be considered as a potential carrier for oral administration.     

High efficiency entrapment of superoxide dismutase into mucoadhesive chitosan-coated liposomes

Superoxide dismutase (SOD), antioxidative enzyme and potential anti-inflammatory agent, was encapsulated into mucoadhesive chitosan-coated liposomes in order to increase its releasing time and to facilitate its cellular penetration. Positively, neutrally and negatively charged liposomes were prepared using soybean lecithin, stearylamine, phosphatidyl glycerol and cholesterol. The effects of liposomal lipid composition and protein to lipid ratio on the encapsulation parameters were studied in three preparation methods: dehydration–rehydration, hydration and proliposome methods. The highest efficiency of SOD entrapment, 39–65%, was achieved by the proliposome method. Vesicles prepared by the hydration method entrapped 1–13% and vesicles prepared by dehydration–rehydration entrapped 2–3% of SOD. Stability tests for SOD-loaded liposomes prepared by the proliposome method showed no significant loss of the enzyme activity within 1 month at 4 °C or within 2 days at 37 °C. Positively, neutrally and negatively charged liposomes, prepared by the proliposome method, were successfully coated with two types of low and medium molecular weight chitosans. Both types of chitosan coating increased the mucoadhesive characteristics of all three types of vesicles. Using the proliposome method and subsequent chitosan coating, highly efficient SOD-loaded vesicles for drug targeting on mucosal tissues could be produced.     

Preparation and characterization of liposomes encapsulating chitosan nanoparticles

Liposomes are an important colloidal carrier system for controlled drug delivery. However some highly hydrophilic small molecules are difficult to entrap into liposomes and store stably, resulting in poor encapsulation efficiency and fast leakage. In the present work, fluorescein sodium (FS) was used as a model drug that was loaded into chitosan nanoparticles and then encapsulated into liposomes by reverse-phase evaporation (RPV). The encapsulation efficiency, particle size, zeta potential, release in vitro and pharmacokinetics in rats were determined in order to characterize the novel drug delivery system. The entrapment efficiency was above 80% in nanoparticles (Np) and 95% in liposomes encapsulating the nanoparticles (Lip-Np). The Lip-Np was composed of soybean phospholipids, cholesterol and chitosan, which the average diameter was 202.6 nm and zeta potential was -34.8 mV. The release rate of fluorescein sodium from Lip-Np was slower than from Np and liposomes. FS in Lip-Np administered to rats exhibited prolonged circulation and higher bioavailability than FS in Np. The results indicated that liposomal release kinetics can be controlled by encapsulating nanoparticles and thus solid-cored liposomes can be used as a potential drug delivery system.